血管内皮生长因子抑制剂生物学活性检测方法的建立

目的建立血管内皮生长因子抑制剂(VEGF Trap)生物学活性检测方法。方法利用HEK293/D9/Flt-18R(al-pha)/Flt-IL18R(beta),clone V3H9细胞系,通过荧光素酶检测系统(Steady-Glo~ Luciferase Assaysystem)进行VEGF Trap的生物学活性检测,用VEGF Trap参考品计算供试品的相对百分效价,并对该方法进行精密性和准确性验证。结果VEGF Trap供试品及参考品在该方法中均存在量效关系,且符合四参数方程式:y=(A-D)/[1+(X/C)B]+D。3批VEGF Trap原液和6批成品经3次测定,相对百分效价的平均值在(90.00±2.40)%～(116.77±16.50)%之间,变异系数均小于15%。1批VEGF Trap原液及成品经3次测定,回收率分别为(90.40±2.67)%和(117.20±18.12)%。结论已成功建立VEGF Trap生物学活性检测方法,该方法重复性好,准确性高,可作为VEGFTrap生物学活性的常规检测方法。     

重组人干扰素β1a生物学活性MTS/PMS检测方法的建立

目的建立重组人干扰素β1a(Recombimant human interferon beta1a,rhIFNβ1a)生物学活性MTS/PMS检测方法。方法将MTS和PMS偶联作为染色液,建立IFNβ1a生物学活性检测方法,对细胞浓度、细胞病变时间、MTS工作浓度和染色时间进行优化,并绘制效应曲线。对建立的方法进行重复性、准确性验证,并与结晶紫染色法进行比较。结果优化后的MTS/PMS法的最佳反应条件为:细胞浓度1×105个/ml,细胞病变时间24～36 h,MTS工作浓度2 mg/ml,染色时间40 min;A570/630值与IFNβ1a保护Wish细胞S型效应曲线的相关系数(R2)均达0.99以上。不同检测板、相同加样位置的变异系数在6%～20%之间;相同检测板、不同加样位置的变异系数在13%～17%之间;检测IFNβ1a细胞收集液的回收率在86%～121%之间。该法检测rhIFNβ1a生物学活性效应曲线呈反"S"型,线型较好,R2值均在0.99以上,均比结晶紫染色法的R2值高,且比结晶紫染色法更稳定。结论已建立了rhIFNβ1a生物学活性MTS/PMS检测方法,适用于常规定量测定rhIFNβ1a的生物学活性。     

重组人干扰素β1b活性测定国家标准品的研制

目的研制重组人干扰素β1b(rhIFNβ1b)生物学活性测定国家标准品。方法按《中国药典》三部(2005版)要求检测rhIFNβ1b标准品原液各项质量指标,以rhIFNβ1b国际标准品为标准进行协作标定,并检测其稳定性。结果rhIFNβ1b国家标准品经检测,外观、无菌试验合格,水分含量为0.8%,分装精度为0.44%。该标准品经3家实验室协作标定24次,几何平均生物学活性为7.18×104IU/支,平均生物学活性的95%可信区间为6.87×104～7.52×104IU/支,单次测定的95%参考值范围为5.76×104～8.68×104IU/支,平均可信限率为4.36%。在温度为-20、4、25和37℃条件下放置12个月,其生物学活性保持稳定。结论该批rhIFNβ1b活性测定国家标准品各项指标均符合要求,可作为国家标准品使用,生物学活性定为7.2×104IU/支,批号为08/01。     

高效液相色谱法检测破伤风抗毒素注射液中苯酚残留量

目的采用高效液相色谱法检测破伤风抗毒素注射液中苯酚残留量,并对方法进行验证及初步应用。方法色谱柱:C18色谱柱;流动相:甲醇-超纯水(体积比1∶1);流速:0.5 ml/min;检测波长:270 nm;柱温:35℃;进样量:5μl。对方法进行专属性、线性、检测限、准确性、精密性验证。用验证后的方法检测4批破伤风抗毒素注射液的苯酚残留量。结果供试品溶液中加入60μg/ml的苯酚对照品溶液和间甲酚对照品溶液的平均回收率为99.92%,相对标准偏差(relative standard deviation,RSD)为0.139 3%;在1~60μg/ml范围内,苯酚对照品色谱峰面积与浓度呈良好的线性关系,回归方程:Y=9.869 19 X+1.082 34 e-1,r=0.999 96;按照信噪比(S/N)≥3确定最低检测限为0.05μg/ml;供试品溶液中加入不同浓度苯酚对照品溶液的平均回收率分别为101.75%和97.03%,RSD分别为0.025%和0.796%;供试品溶液5次检测结果的RSD为1.485 9%。4批破伤风抗毒素注射液中的苯酚残留量均符合《中国药典》三部(2010版)≤890μg/ml的要求。结论高效液相色谱法检测破伤风抗毒素注射液中苯酚残留量快速、简便,且专属性、线性、准确性、精密性良好。     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白受体结合活性检测方法的改进

目的对重组人Ⅱ型肿瘤坏死因子受体(Tumor necrosis factor receptor,TNFR)-抗体融合蛋白的受体结合活性检测方法进行改进。方法采用夹心ELISA法对系列稀释的重组人Ⅱ型TNFR-抗体融合蛋白供试品和参比品进行检测,通过四参数方程拟合,计算二者的半数有效浓度(EC50),分析其受体结合活性。对改进的方法进行精密性和准确性验证,并与原方法进行比较。结果重组人Ⅱ型TNFR-抗体融合蛋白供试品和参比品均存在量效关系,符合四参数方程y=(A-D)/[1+(X/C)B]+D;3批重组人Ⅱ型TNFR-抗体融合蛋白经3次测定,受体结合活性分别为(133±8)、(114±4)和(138±9)BU,符合其质量标准中受体结合活性65～135 BU的规定,变异系数分别为6.02%、3.51%和6.52%;1批供试品经3次重复测定,回收率为(107.67±7.50)%;3批供试品采用原方法和改进方法分别重复测定3次,受体结合活性差异均无统计学意义(P>0.05)。结论改进的方法精密性好,准确性高,可作为重组人Ⅱ型TNFR-抗体融合蛋白受体结合活性的常规检测方法。     

抗CD20单克隆抗体质量控制中生物学活性的趋势分析

目的对抗CD20单克隆抗体质量控制中的生物学活性进行趋势分析。方法利用人淋巴瘤Raji细胞株作为靶细胞,通过补体依赖的细胞毒作用(complement dependent cytotoxicity,CDC),使用Cyto Tox 96 Non-Radioactive Cytotoxicity Assay试剂监测抗CD20单抗的生物学活性,以抗CD20单抗参比品计算供试品的相对百分效价。通过对中国食品药品检定研究院(NIFDC)和企业质控实验室99批次抗CD20单抗生物学活性的检测结果,分别建立警戒限(均值±2 SD)和行动限(均值±3 SD),绘制趋势分析图,并对检测结果进行连续性及周期性趋势分析。结果对2009~2013年某企业抗CD20单抗的生物学活性测定结果显示,抗CD20单抗供试品和参比品在CDC活性中均存在量效关系,且符合四参数方程:y=(A-D)/[1+(X/C)B]+D。NIFDC体外CDC活性的相对百分效价为(99.95±10.83)%,企业自检的相对百分效价为(100.71±9.29)%;对上述结果进行连续性趋势分析,未发现超出行动限的结果;对不同年度间数据进行方差分析显示,年度间差异无统计学意义(P0.05),数据具有可比性;对年度数据进行周期性趋势分析,结果均在行动限以内,无过高或过低结果,未出现严重的漂移或连续两批结果相差4 SD以上结果,总体趋势较平稳。结论首次通过对抗CD20单克隆抗体生物学活性的趋势分析,反映了该制品的批间一致性以及生产工艺的稳定性,也为其他单抗类治疗药物质量控制中关键质量属性的趋势分析提供了参考。     

破伤风抗毒素效价絮状单位测定方法(体外法)的验证及其与体内法的比较

目的对破伤风抗毒素絮状单位测定法(体外法)进行方法验证,并与小鼠中和试验法(体内法)进行比较。方法对用于破伤风抗毒素效价检测的絮状单位测定法进行准确度、精密度、专属性及耐用性验证。采用絮状单位测定法及小鼠中和试验法分别检测破伤风抗毒素原料血浆、原液及成品,并分析两种方法的相关性。结果对破伤风抗毒素国家标准品6次絮状测定回收率均为100%;重复性验证的变异系数(CV)均2%;对破伤风抗毒素效价检测具有良好的专属性,供试品中防腐剂对其检测无干扰;不同温度下检测结果的CV值2%。两种方法对原料血浆、原液及成品测定结果的相关系数分别为0.825、0.937及0.955,P均0.001,呈正相关,且相关性显著。结论破伤风抗毒素絮状单位测定法(体外法)操作简便,具有良好的准确性、精密性、专属性及耐用性,且与体内法具有显著相关性,可作为企业内控的一种辅助快速检测手段。     

重组人源化抗CD52单克隆抗体相对抗原结合活性检测方法的建立及验证

目的建立重组人源化抗CD52单克隆抗体相对抗原结合活性的检测方法,并进行验证。方法测定3种人淋巴瘤细胞(MC/CAR、HUT78、RAMOS)CD52抗原的表达率,选择CD52表达率最高的作为靶细胞。以系列稀释的重组人源化抗CD52单克隆抗体的参比品和供试品作用表达CD52抗原的人淋巴瘤细胞,FITC标记的兔抗人Ig G结合人淋巴瘤细胞上的重组人源化抗CD52单克隆抗体,流式细胞分析仪测定几何荧光均数。通过四参数方程拟合,计算参比品和供试品的半数有效浓度(EC50)及相对结合活性。并对该方法的专属性、准确性、精密性、线性范围和耐用性进行验证。结果淋巴瘤MC/CAR细胞CD52抗原表达率最高,确定该细胞为靶细胞。CD52抗原与无关抗体不存在特异性结合;重复3次测定不同理论相对结合活性人源化抗CD52单克隆抗体参比品的回收率在97.4%~111.9%之间,相对结合活性及回收率的RSD值均在10%以内;同一实验人员于同一天6次重复检测重组人源化抗CD52单克隆抗体相对结合活性在86.93%~114.42%之间,RSD值在10%以内,3 d内不同实验员分别检测5个效价样品的相对结合活性的RSD在20%以内;在重组人源化抗CD52单克隆抗体理论效价50%~150%范围内,与相对结合活性呈良好的线性,线性回归方程为y=1.045 2 x-1.082,R2为0.992 1;MC/CAR细胞在第28代时仍适用于抗CD52抗体相对结合活性的测定。结论该方法具有良好的特异性、准确性、精密性、线性和耐用性,且操作简便,可用于重组抗CD52抗体抗原结合活性的常规检测。     

前蛋白转化酶枯草溶菌素9单克隆抗体抗原结合活性间接ELISA测定方法的建立

目的建立前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)单克隆抗体抗原结合活性的间接ELISA测定方法。方法利用链酶亲和素-生物素系统捕获包被PCSK9蛋白,采用间接ELISA法检测PCSK9单抗参比品和供试品,通过拟合四参数方程Y=(A-D)/[1+(X/C)~B]+D,得出两者的半数有效浓度(concentration for 50%of maximal effect,EC_(50)),计算供试品的抗原结合活性。同时验证该方法的专属性和精密性。结果 PCSK9单抗参比品、供试品及Evolocumab(Amgen)的曲线均呈显著的剂量依赖关系,符合四参数方程,且R~20.99;抗CD115抗体及空白稀释液不存在类似的剂量效应曲线;重复性、人员及日间精密性的相对标准偏差(relative standard deviatin,RSD)分别为7.5%、11.3%和10.4%,均15%。结论成功建立了抗PCSK9单抗抗原结合活性的间接ELISA检测方法,该方法专属性和精密度良好,可用于PCSK9单抗抗原结合活性的常规测定。     

前蛋白转化酶枯草溶菌素9单克隆抗体生物学活性荧光染料标记法的建立、优化及验证

目的建立并优化前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)单克隆抗体生物学活性的荧光染料标记检测法,并进行验证。方法对建立的荧光染料标记法的细胞铺板密度(1×10~5、5×10~5和1×10~6个/mL)、PCSK9蛋白浓度(5、20、40、80μg/mL)、荧光标记的低密度脂蛋白(low density lipoprotein labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate,Dil-LDL)浓度(10、16、20μg/mL)、单克隆抗体的浓度梯度(200、52、20、12、8、4、0. 8μg/mL,200、30、10、6、4、1、0. 2μg/mL,200、28、9、6、4、2、0. 2μg/mL)进行优化,并对优化方法的专属性、准确度、精密度、线性范围、耐用性进行验证。结果最适方法检测条件:铺板密度为5×10~5个/mL,PCSK9蛋白和Dil-LDL浓度分别为20和16μg/mL,单克隆抗体的浓度梯度为200、52、20、12、8、4、0. 8μg/mL。该方法可特异性检测PCSK9单克隆抗体;供试品检测回收率在70%～130%之间;供试品检测结果的相对标准偏差(RSD)均10%;供试品效价在50%～150%之间时,检测值与理论值呈良好的线性关系,线性拟合方程为y=1. 002 x-0. 006 7,R~2=0. 997 8;耐用性检测活性均在70%～140%范围内。结论本实验建立的方法具有良好的专属性、准确度和精密度,可用于PCSK9单克隆抗体的生物学活性测定。     

The Wnt Signaling Pathway Inhibitors Improve the Therapeutic Activity of Glycolysis Modulators against Tongue Cancer Cells

Excessive glucose metabolism and disruptions in Wnt signaling are important molecular changes present in oral cancer cells. The aim of this study was to evaluate the effects of the combinatorial use of glycolysis and Wnt signaling inhibitors on viability, cytotoxicity, apoptosis induction, cell cycle distribution and the glycolytic activity of tongue carcinoma cells. CAL 27, SCC-25 and BICR 22 tongue cancer cell lines were used. Cells were treated with inhibitors of glycolysis (2-deoxyglucose and lonidamine) and of Wnt signaling (PRI-724 and IWP-O1). The effects of the compounds on cell viability and cytotoxicity were evaluated with MTS and CellTox Green tests, respectively. Apoptosis was evaluated by MitoPotential Dye staining and cell cycle distribution by staining with propidium iodide, followed by flow cytometric cell analysis. Glucose and lactate concentrations in a culture medium were evaluated luminometrically. Combinations of 2-deoxyglucose and lonidamine with Wnt pathway inhibitors were similarly effective in the impairment of oral cancer cells’ survival. However, the inhibition of the canonical Wnt pathway by PRI-724 was more beneficial, based on the glycolytic activity of the cells. The results point to the therapeutic potential of the combination of low concentrations of glycolytic modulators with Wnt pathway inhibitors in oral cancer cells.     

洗洁精对人皮肤成纤维细胞损伤刺激的研究

借助人体皮肤纤维细胞损伤刺激实验,通过MTT显色图和细胞结晶紫染色图分析了洗洁精对人体皮肤的影响。     

应用免疫印迹法检测人基因工程干扰素中大肠杆菌残余成分

用大肠杆菌抽提物免疫家兔制备相应的抗血清,并用该血清建立免疫印迹法,检测基因工程干扰素中大肠杆菌残余成分。初步试验结果表明,该方法的灵敏度高于SDS-PAGE银染法。检测3批人α_1.型基因工程干扰素的结果表明,制品的纯度较好。     

rHu-TPO体外生物学活性测定方法的建立

应用TPO依赖细胞株检测rHu TPO体外生物学活性。将TPO标准品和样品分别稀释到一定的浓度 ,加入到 96孔细胞培养板中 ,每孔加入TPO依赖细胞 ,在 37℃CO2 孵箱中培养 48h后 ,加入一种四唑化合物 ,再培养6± 2h ,在 492nm波长下测A值。用TPO标准品做对照。根据剂量反应平行线法 ,计算出样品相对的体外生物学活性。该方法重复性好 ,回收率高 ,便于推广应用     

One-Pot Three-Component Synthesis of Novel Diethyl((2-oxo-1,2-dihydroquinolin-3-yl)(arylamino)methyl)phosphonate as Potential Anticancer Agents

With the aim of discovering new anticancer agents, we have designed and synthesized novel α-aminophosphonate derivatives containing a 2-oxoquinoline structure using a convenient one-pot three-component method. The newly synthesized compounds were evaluated for antitumor activities against the A549 (human lung adenocarcinoma cell), HeLa (human cervical carcinoma cell), MCF-7 (human breast cancer cell), and U2OS (human osteosarcoma cell) cancer cell lines in vitro, employing a standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The results of pharmacological screening indicated that many compounds exhibited moderate to high levels of antitumor activities against the tested cancer cell lines and that most compounds showed more potent inhibitory activities comparable to 5-fluorouracil (5-FU) which was used as a positive control. The mechanism of representative compound 4u (diethyl((2-oxo-1,2-dihydroquinolin-3-yl)(phenyl-amino)methyl)phosphonate) indicated that the compound mainly arrested HeLa cells in S and G2 stages and was accompanied by apoptosis in HeLa cells. This action was confirmed by acridine orange/ethidium bromide staining, Hoechst 33342 staining, and flow cytometry.     

Preparation of micro-crystal stones from molten titanium slag by “one-step” cooling method

Medium-titanium blast furnace slag (MTS) is a major solid waste in the smelting process of vanadium and titanium magnetite. The moderate TiO₂ content in MTS makes it unsuitable for titanium extraction and difficult to be used as cement mixing material in large quantities. In this study, the molten MTS was utilized to prepare micro-crystal stones by a “one-step” cooling method, and the TiO₂ in the slag was used as a nucleating agent. The effects of the cooling process and MTS utilization on the slag structure, crystallization characteristics, and physical properties of the micro-crystal stones were investigated. The results show that when the utilization of MTS is 100 wt%, the main crystalline phase of the micro-crystal stones is akermanite, the compressive strength and micro-hardness are 142.5 MPa and 696.9 kgf·mm⁻². When the utilization of MTS is 90 wt%, the main phases of microcrystalline stone are augite and akermanite, and the compressive strength rises to 194.8 MPa. Hence, through proper cooling control, the preparation of micro-crystal stones with good performance by using molten MTS as raw material will have considerable economic value.     

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O₂) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.     

Numerical Simulation of Effect of Methyltrichlorosilane Flux on Isothermal Chemical Vapor Infiltration Process of C/SiC Composites

A two-dimensional axisymmetrical mathematical model for the isothermal chemical vapor infiltration process of C/SiC composites was developed. Transport phenomena of momentum, energy, and mass in conjunction with infiltration-induced changes of preform structure were taken into account. The integrated model was implemented by the finite-element method to simulate numerically the isothermal chemical vapor infiltration (ICVI) process of C/SiC composites at different methyltrichlorosilane (MTS) fluxes. The influence of MTS flux on concentration distribution and time-dependent densification behaviors of C/SiC composites was studied in detail. Calculation results imply that MTS flux has an obvious influence on infiltration in micro-pores and little influence on infiltration in macro-pores. Increasing flux will lead to an evident acceleration for infiltration in micro-pores. Moderate flux is preferable by a combination of both a relatively high infiltration rate and a relatively low fabrication cost. This model is helpful to understand the fundamentals of the ICVI process for the fabrication of C/SiC composites.     

Herbally derived polymeric nanofibrous scaffolds for bone tissue regeneration

Hydroxyapatite (HA), the bone mineral and Cissus quadrangularis (CQ), a medicinal plant with osteogenic activity, are attaining increasing interest as a potential therapeutic agent for enhanced bone tissue regeneration. In the present study a synergistic effect of these two agents were analyzed by fabricating PCL‐CQ‐HA nanofibrous scaffolds by electrospinning and compared with PCL‐CQ and PCL (control) nanofibrous scaffolds. Morphology, composition, hydrophilicity, and mechanical properties of the electrospun PCL, PCL‐CQ, PCL‐CQ‐HA nanofibrous scaffolds were examined by Field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), Contact angle and Tensile tests, respectively. The response of human foetal osteoblast cells on these scaffolds were evaluated using MTS assay, alkaline phosphatase activity, alizarin red staining, and osteocalcin expression for bone tissue regeneration. While the observed cellular response to both groups of scaffolds was better than for the control PCL scaffold, the PCL‐CQ‐HA nanofibrous scaffolds provided the most favorable substrate for cell proliferation and mineralization. The results showed that PCL‐CQ‐HA nanofibrous scaffolds had appropriate surface roughness for the osteoblast adhesion, proliferation, and mineralization comparing with other scaffolds. The observed investigation of physicochemical and biological properties suggests that the CQ‐HA loaded PCL nanofibrous scaffolds serve as a potential biocomposite material for bone tissue engineering. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39835.